U.S. patent number 11,448,646 [Application Number 15/869,146] was granted by the patent office on 2022-09-20 for isolating a target analyte from a body fluid.
This patent grant is currently assigned to DNAE Group Holdings Limited. The grantee listed for this patent is DNAE Group Holdings Limited. Invention is credited to Eddie W. Adams, Lisa-Jo Ann Clarizia, Sergey A. Dryga.
United States Patent |
11,448,646 |
Clarizia , et al. |
September 20, 2022 |
Isolating a target analyte from a body fluid
Abstract
The invention generally relates to using magnetic particles and
magnets to isolate a target analyte from a body fluid sample. In
certain embodiments, methods of the invention involve introducing
magnetic particles including a target-specific binding moiety to a
body fluid sample in order to create a mixture, incubating the
mixture to allow the particles to bind to a target, applying a
magnetic field to capture target/magnetic particle complexes on a
surface, and washing with a wash solution that reduces particle
aggregation, thereby isolating target/magnetic particle
complexes.
Inventors: |
Clarizia; Lisa-Jo Ann
(Albuquerque, NM), Adams; Eddie W. (Albuquerque, NM),
Dryga; Sergey A. (Albuquerque, NM) |
Applicant: |
Name |
City |
State |
Country |
Type |
DNAE Group Holdings Limited |
London |
N/A |
GB |
|
|
Assignee: |
DNAE Group Holdings Limited
(London, GB)
|
Family
ID: |
1000006570965 |
Appl.
No.: |
15/869,146 |
Filed: |
January 12, 2018 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180149642 A1 |
May 31, 2018 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
12850203 |
Aug 4, 2010 |
|
|
|
|
61326588 |
Apr 21, 2010 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/54333 (20130101); G01N 33/56911 (20130101); G01N
33/54326 (20130101); B01L 2200/0647 (20130101); G01N
2333/195 (20130101); C07K 16/1267 (20130101); G01N
2446/20 (20130101); B01L 2400/043 (20130101); C12Q
1/689 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); G01N 33/569 (20060101); C07K
16/12 (20060101); C12Q 1/689 (20180101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
2 342 047 |
|
Sep 2001 |
|
CA |
|
2138835 |
|
Dec 2009 |
|
EP |
|
2261650 |
|
Dec 2010 |
|
EP |
|
1988/003957 |
|
Jun 1988 |
|
WO |
|
39/06699 |
|
Jul 1989 |
|
WO |
|
90/08841 |
|
Aug 1990 |
|
WO |
|
91/02811 |
|
Mar 1991 |
|
WO |
|
92/08805 |
|
May 1992 |
|
WO |
|
92/15883 |
|
Sep 1992 |
|
WO |
|
92/17609 |
|
Oct 1992 |
|
WO |
|
95/31481 |
|
Nov 1995 |
|
WO |
|
98/20148 |
|
May 1998 |
|
WO |
|
99/53320 |
|
Oct 1999 |
|
WO |
|
01/73460 |
|
Oct 2001 |
|
WO |
|
02/98364 |
|
Dec 2002 |
|
WO |
|
2005/026762 |
|
Mar 2005 |
|
WO |
|
2005106480 |
|
Nov 2005 |
|
WO |
|
2007/018601 |
|
Feb 2007 |
|
WO |
|
2007/123345 |
|
Nov 2007 |
|
WO |
|
2007/135099 |
|
Nov 2007 |
|
WO |
|
2007123342 |
|
Nov 2007 |
|
WO |
|
2008/119054 |
|
Oct 2008 |
|
WO |
|
2008/139419 |
|
Nov 2008 |
|
WO |
|
2008147530 |
|
Dec 2008 |
|
WO |
|
2009/048673 |
|
Apr 2009 |
|
WO |
|
2009/055587 |
|
Apr 2009 |
|
WO |
|
2009072003 |
|
Jun 2009 |
|
WO |
|
2009/122216 |
|
Oct 2009 |
|
WO |
|
2010036827 |
|
Apr 2010 |
|
WO |
|
WO-2010123594 |
|
Oct 2010 |
|
WO |
|
2011/019874 |
|
Feb 2011 |
|
WO |
|
2011/133630 |
|
Oct 2011 |
|
WO |
|
2011/133632 |
|
Oct 2011 |
|
WO |
|
2011/133759 |
|
Oct 2011 |
|
WO |
|
2011/133760 |
|
Oct 2011 |
|
WO |
|
2009155384 |
|
Dec 2020 |
|
WO |
|
Other References
Sigma, "Tris(hydroxymethyl)aminomethane; Tris", Technical Bulletin
No. 106B, 1996, retrieved from
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulleti-
n/1/106bbul.pdf on Sep. 5, 2016 (Year: 1996). cited by examiner
.
Science Encyclopedia, "Osmosis (Cellular)--Osmosis in red blood
cells", 2009, retrieved from
https://web.archive.org/web/20090727133453/https://science.jrank.org/page-
s/4930/Osmosis-Cellular-Osmosis-in-red-blood-cells.html on Sep. 11,
2021 (Year: 2009 ). cited by examiner .
Cross, et al., Choice of Bacteria in Animal Models of Sepsis,
Infec. Immun. 61(7):2741-2747 (1983). cited by applicant .
Dam et al. "Garlic (Allium sativum) Lectins Bind to High Mannose
Oligosaccharide Chains", Journal of Biological Chemistry vol. 273,
No. 10, Issue of March 6, pp. 5528-5535, 1998. cited by applicant
.
Djukovic, et al., Signal Enhancement in HPLC/Microcoil NMR Using
Automated Column Trapping, Anal. Chem., 78:7154-7160 (2006). cited
by applicant .
Dover, Jason E., et al. "Recent advances in peptide probe-based
biosensors for detection of infectious agents." Journal of
microbiological methods 78.1 (2009): 10-19. cited by applicant
.
Drancourt, et al., Diagnosis of Mediterranean Spotted Fever by
Indirect Immunofluorescence of Rickettsia conorii in Circulating
Endothelial Cells Isolated with Monoclonal Antibody-Coated
Immunomagnetic Beads, J. Infectious Diseases, 166(3):660-663, 1992.
cited by applicant .
Dynabeads.RTM. for Immunoassay IVD, retrieved from http://www.in
vitrogen.com/site/i3s/en/home/Products-and-Services/Applications/Diagnost-
icsClinical-Research/Bead-based-IVD-Assavs/Bead-based-Immunoassav-iVD.html
on May 29, 2013, four pages). cited by applicant .
Elnifro, Elfath M., et al. "Multiplex PCR: optimization and
application in diagnostic virology." Clinical Microbiology Reviews
13.4 (2000): 559-570. cited by applicant .
Engvall, Enzyme immunoassay ELISA and EMIT, Meth. in Enzymol.,
70:419-439 (1980). cited by applicant .
Extended European Search Report dated Aug. 20, 2014 in EP
11864030.9. cited by applicant .
Extended European Search Report dated Feb. 2, 2017 in EP
16190239.0. cited by applicant .
Extended European Search Report, dated Oct. 15, 2013 in EP
11772606.7. cited by applicant .
Fan, et al., Self-assembly of ordered, robust, three-dimensional
gold nanocrystal/silica arrays, Science, 304:567 (2004). cited by
applicant .
Fenwick et al., 1986, Mechanisms Involved in Protection Provided by
Immunization against Core Lipopolysaccarides of Escherichia coli J5
from Lethal Haemophilus pleuropneumoniae Infections in Swine,
Infection and Immunity 53 (2):298-304. cited by applicant .
Fu, et al., Rapid Detection of Escherichia coli O157:H7 by
Immunogmagnetic Separation and Real-time PCR, Int. J. Food
Microbiology, 99(1):47-57, (2005). cited by applicant .
Fung, M-C., et al. PCR amplification of mRNA directly from a crude
cell lysate prepared by thermophilic protease digestion. Nucleic
Acids Research, vol. 19 (15), p. 4300, 1991. cited by applicant
.
Furdui, Vasile I. et al., "Immunomagnetic T Cell Capture From Blood
for PCR Analysis Using Microfluidic Systems", Lab on a Chip, 2004,
vol. 4 No. 6, pp. 614-618 (5 Pages). cited by applicant .
Furdui, Vasile I. et al., "Microfabricated Electrolysis Pump System
for Isolating Rare Cells in Blood; Micro-Electrolysis Pumps for
Blood", Journal of Micromechanics & Microengineering, vol. 13,
No. 4, Jul. 1, 2003, pp. S164-S170 (7 Pages). cited by applicant
.
Gesbert et al. "Asparagine Assimilation is Critical for
Intracellular Replication and Dissemination of Francisella"
Cellular Microbiology, 2014, 16(3), pp. 434-449 (16 Pages). cited
by applicant .
Goding, J.W., Conjugation of antibodies with fluorochromes:
modifications to the standard methods, J. Immunol. Meth., 13:215
(1976). cited by applicant .
Goloshevsky, et al., Development of Low Field Nuclear Magnetic
Resonance Microcoils, Rev. Sci. Inst.., 76:024101-1 to 024101-6
(2005). cited by applicant .
Grant, et al., Analysis of Multilayer Radio Frequency Microcoils
for Nuclear Magnetic Resonance Spectroscopy, IEEE Trans. Magn.,
37:2989-2998 (2001). cited by applicant .
Grant, et al., NMR Spectroscopy of Single Neurons, Magn. Reson.
Med., 44:19-22 (2000). cited by applicant .
Giiflilhs et al., 1994, Isolation of high affinity human antibodies
directly from large synthetic repertoires. EMBO J. 13
(14):3245-3260. cited by applicant .
Gu et al., 2003, Using Biofunctional Magentic Nanoparticles to
Capture Vancomycin-Resistant Enterococci and Other Gram-Positive
Bacteria at Ultralow Concentration, J. Am. Chem. Soc.,
125:15702-15703. cited by applicant .
Gu et al., 2006, Biofunctional magnetic nanoparticles for protein
separation and pathogen detection, Chem. Commun.:941-949. cited by
applicant .
Halbach, Design of Permanent Multipole Magnets with Oriented Rare
Earth Cobalt Material, Nuclear Instrum Methods, 169:1-10 (1980).
cited by applicant .
Harada, et al., Monoclonal antibody G6K12 specific for
membrane-associated differentiation marker of human stratified
squamous epithelia and squamous cell carcinoma, J. Oral. Pathol.
Med., 22(4):1145-152 (1993). cited by applicant .
Harkins and Harrigan, "Labeling of Bacterial Pathogens for Flow
Cytometric Detection and Enumeration" Curr Prot Cytom (2004)
11.17.1-11.17.20. cited by applicant .
Harlow, et al., 1988, 'Antibodies', Cold Spring Harbor Laboratory,
pp. 93-117. cited by applicant .
Harris et al., Science 320:106-109 (2008). cited by applicant .
Heijnen et al., 2009, Method for rapid detection of viable
Escherichia coli in water using real-time NASBA, Water Research,
43:3124-3132. cited by applicant .
Hijmans, et al., An immunofluorescence procedure for the detection
of intracellular immunoglobulins, Clin. Exp. Immunol., 4:457
(1969). cited by applicant .
Hirsch, et al., Easily reversible desthiobiotin binding to
streptavidin, avidin, and other biotin-binding proteins: uses for
protein labeling, detection, and isolation, Anal. Biochem.,
208(2):343-57 (2002). cited by applicant .
Hongwei Gu et al:: Using Biofunctional Magnetic Nanoparticles to
Capture Vancomycin-Resistent Enterococci and Other Gram-Positive
Bacteria at Ultralow Concentration, Journal of the American
Chemical Society, vol. 125, No. 51, Dec. 1, 2003 (Dec. 1, 2003),
pp. 15702-15703, XP055087066, ISSN; 002-7863, DOI:
10.1021/ja0359310. cited by applicant .
Hoult and Richards, The Signal-to-Noise Ratio of the Nuclear
Magnetic Resonance Experiment, J. Magn. Reson., 24:71-85 (1976).
cited by applicant .
Hunter, et al., Immunoassays for Clinical Chemistry, pp. 147-162,
Churchill Livingston, Edinborough (1983). cited by applicant .
Inai, et al., Immunohistochemical detection of an enamel
protein-related epitope in rat bone at an early stage of
osteogenesis. Histochemistry, 99(5):335-362 (1993). cited by
applicant .
International Search Report dated Feb. 27, 2014 for
PCT/US2013/076649. cited by applicant .
International Search Report dated Jul. 25, 2011 for
PCT/US2011/33184. cited by applicant .
International Search Report dated Jun. 22, 2011 for
PCT/US2011/33186. cited by applicant .
International Search Report dated Jul. 19, 2011 for
PCT/US2011/33410. cited by applicant .
International Search Report dated Jun. 22, 2011 for
PCT/US2011/33411. cited by applicant .
ISR and Written Opinion dated Oct. 29, 2008 for PCT/US2008/062473.
cited by applicant .
ISR and Written Opinion dated Mar. 3, 2009 for PCT/US2008/080983.
cited by applicant .
ISR and Written Opinion dated Feb. 5, 2010 for PCT/US2009/067577.
cited by applicant .
ISR and Written Opinion dated Dec. 22, 2011 for PCT/US2011/48447.
cited by applicant .
ISR and Written Opinion dated Dec. 22, 2011 for PCT/US2011/48452.
cited by applicant .
ISR and Written Opinion dated Jul. 7, 2008 for PCT/US2008/058518.
cited by applicant .
IPRP dated Jul. 7, 2008 for PCT/US2008/058518. cited by applicant
.
Cooper et al., 2011, A micromagnetic flux concentrator device for
isolation and visualization of pathogens. 15th International
Conference on Miniaturized Systems for Chemistry and Life Sciences.
Oct. 2-6, 2011, Seattle, Washington, USA. cited by applicant .
Johnson, Thermal Agitation of Electricity in Conductors, Phys.
Rev., 32:97-109 (1928). cited by applicant .
Kaittanis, et al., One-step nanoparticle mediated bacterial
detection with magentic relaxation, Nano Lett., 7(2):381-383
(2007). cited by applicant .
Klaschik, S., L. E. Lehmann, et al. (2002). "Real-time PCR for
detection and differentiation of gram-positive and gramnegative
bacteria." J Clin Microbiol 40(11): 4304-4307. cited by applicant
.
Lecomte et al. Nucl Acids Res. 11.7505 (1983) "Selective
Inactivation of the 3' to 5' exonuclease activity of Escherichia
coli DNA bolymerase I by heat". cited by applicant .
Lee, et al., Chip-NMR Biosensor for detection and molecular
analysis of cells, Nature Medicine, 14(8):869-874 (2008). cited by
applicant .
Levin, Cell 88:5-8(1997). cited by applicant .
Li et al., 2010, Chemiluminescent Detect of E. coli O157:H7 Using
Immunological Method Based on Magnetic Nanoparticles, J. of
Nanoscience and Nanotechnology 10:696-701. cited by applicant .
Lu et al., 2007, Magnetic Nanoparticles: Synthesis, Protection,
Functionalization, and Application, Angew. Chem. Int. Ed.
46:1222-1244. cited by applicant .
Lund, et al., Immunomagnetic separation and DNA hybridization for
detection of enterotoxigenic Escherichia coli in a piglet model, J
Clin. Microbiol., 29:2259-2262 (1991). cited by applicant .
Madonna A J, et al. "Detection of Bacteria from Biological Mixtures
Using Immunomagnetic Separation Combined with Matrix-Assisted Laser
Desorption/Ilonization Time-of-flight Mass Spectrometry", Rapid
Communications in Mass Spectrometry, John WIley & Sons, GB,
vol. 15, No. 13, Jan. 1, 2001, pp. 1068-1074. cited by applicant
.
Magin, et al., Miniature Magnetic Resonance Machines, IEEE Spectrum
34(10):51-61 (1997). cited by applicant .
Malba, et al., Laser-lathe Lithography--A Novel Method for
Manufacturing Nuclear Magnetic Resonance Microcoils, Biomed.
Microdev., 5:21-27 (2003). cited by applicant .
Margin, et al., High resolution microcoil 1 H-NMR for mass-limited,
nanoliter-volume samples, Science, 270:1967 (1995). cited by
applicant .
Margulies et al., Nature, 437: 376-380 (2005). cited by applicant
.
Massin, et al., Planar Microcoil-based magnetic resonance imaging
of cells, Transducers '03, The 12th Int. Conf. on Solid State
Sensors, Actuators, and Microsystems, Boston, Jun. 8-12, pp.
967-970 (2003). cited by applicant .
Massin, et al., Planar Microcoil-based Microfluidic NMR Probes, J.
Magn. Reson., 164:242-255 (2003). cited by applicant .
Matar et al., 1990, Magnetic particles derived from iron nitride,
IEEE Transactions on magnetics 26(1):60-62. cited by applicant
.
McDowell, et al., Operating Nanoliter Scale NMR Microcoils in a
Itesla Field, J. Mag. Reson., 188(1):74-82 (2007). cited by
applicant .
Minard, et al., Solenoidal Microcoil Design, Part I: Optimizing RF
Homogeneity and coil dimensions, Concepts in Magn. Reson.,
13(2):128-142 (2001). cited by applicant .
Moreira et al., 2008, Detection of Salmonella typhimurium in Raw
Meats using In-House Prepared Monoclonal Antibody Coated Magnetic
Beads and PCR Assay of the fimA Gene. Journal of Immunoassay &
Immunochemistry 29:58-69. cited by applicant .
Moresi and Magin, Miniature Permanent Magnet for Table-top NMR,
Concept. Magn. Res., 19B:35-43 (2003). cited by applicant .
Moudrianakis et al., Proc. Natl. Acad. Sci. 53:564-71 (1965). cited
by applicant .
Mulder, et al., Characterization of two human monoclonal antibodies
reactive with HLA-B12 and HLA-B60, respectively, raised by in vitro
secondary immunization of peripheral blood lymphocytes, Hum.
Immunol., 36 (3):186-192 (1993). cited by applicant .
Myers and Gelfand, Biochemistry 30:7661 (1991). cited by applicant
.
Narang et al., Methods Enzymol., 68:90 (1979). cited by applicant
.
NCBI Geo Gene Expression Omnibus, Entry for GSE22885, retrieved
from https://www.nobi.nlm.nih.gov/geo/query/acc.cgi?aco=GSE22885 on
May 25, 2017, with excerpts of files
GPL10672_IMS_annotation.ann.txt.gz and GSM565264.txt.gz therein
(five pages total), publicly available on Jul. 13, 2010 (5 Pages).
cited by applicant .
Nordstrom et al., J. Biol. Chem. 256:3112 (1981). cited by
applicant .
Nyquist, Thermal Agitation of Electrical Charge in Conductors,
Phys. Rev., 32:110-113 (1928). cited by applicant .
Ohno et al., 2011, Effects of Blood Group Antigen-Binding Adhesin
Expression during Helicobacter pylori Infection of Mongolian
Gerbils, The Journal of Infectious Diseases 203:726-735. cited by
applicant .
DOson, et al., High-resolution microcoil NMR for analysis of
mass-limited, nanoliter samples, Anal. Chem., 70:645-650 (1998).
cited by applicant .
Olsvik, et al., "Magnetic Separation Techniques in Diagnostic
Microbiology," Clinical Microbiol Rev 1994. cited by applicant
.
Pappas, et al., Cellular Separations: A Review of New Challenges in
Analytical Chemistry, Analytica Chimica Acta, 601 (1):26-35 (2007).
cited by applicant .
Payne, M.J. et al., "The Use of Immobilized Lectins in the
Separation of Staphylococcus aureus, Escherichia coli, Listeria and
Salmonella spp. from Pure Cultures and Foods", Journal of Applied
Bacteriology, 1992, No. 73, pp. 41-52 (12 Pages). cited by
applicant .
Peck, et al., Design and Analysis of Microcoils for NMR Microscopy,
J. Magn. Reson. B 108:114-124 (1995). cited by applicant .
Peck, et al., RF Microcoils patterned using microlithographic
techniques for use as microsensors in NMR, Proc. 15th Ann. Int.
Conf. of the IEEE, Oct. 28-31, pp. 174-175 (1993). cited by
applicant .
Perez, et al., Viral-induced self-assembly of magnetic nanoparticle
allows detection of viral particles in biological media, J. Am.
Chem. Soc., 125:10192-10193 (2003). cited by applicant .
Qiu, et al., Immunomagnetic separation and rapid detection of
bacteria using bioluminescence and microfluidics, Talanta,
79:787-795 (2009). cited by applicant .
Rogers, et al., Using microcontact printing to fabricate microcoils
on capillaries for high resolution proton nuclear magnetic
resonance on nanoliter volumes, Appl. Phys. Lett., 70:2464-2466
(1997). cited by applicant .
Safarik et al., "The application of magnetic separations in applied
Microbiology" Journal of Applied Bacteriology 1995, 78, 575-585.
cited by applicant .
Seeber, et al., Design and Testing of high sensitivity
Microreceiver Coil Apparatus for Nuclear Magnetic Resonance and
Imaging, Rev. Sci. Inst., 72:2171-2179 (2001). cited by applicant
.
Seeber, et al., Triaxial Magnetic Field Gradient System for
Microcoil Magnetic Resonance Imaging, Rev. Sci. Inst., 71:4263-4272
(2000). cited by applicant .
Sillerud, et al., 1H NMR Detection of Superparamagnetic
Nanoparticles at 1 T using a Microcoil and Novel Tuning Circuit, J.
Magn. Reson. 181:181-190 (2006). cited by applicant .
Sista et al., 2008, Heterogeneous Immunoassays Using Magnetic beads
On a Digital Microfluidic Platform, Lab Chip 8 (2):21 88-2196.
cited by applicant .
Skjerve, et al., Detection of Listeria monocytogenes in foods by
immunomagnetic separation, Appl. Env. Microbiol., 56:3478 (1990).
cited by applicant .
Soni et al., Clin Chem 53:1996-2001 (2007). cited by applicant
.
Sorli, et al., Micro-spectrometer for NMR: analysis of small
quantities in vitro, Meas. Sci. Technol., 15:877-880 (2004). cited
by applicant .
Stauber, et al., Rapid generation of monoclonal antibody-secreting
hybridomas against African horse sickness virus by in vitro
immunization and the fusion/cloning technique, J. Immunol. Methods,
161(2):157-168 (1993). cited by applicant .
Stenesh and McGowan, Biochim Biophys Acta, 475:32 (1977). cited by
applicant .
Johne, et al., Staphylococcus aureus exopolysaccharide in vivo
demonstrated by immunomagnetic separation and electron microscopy,
J. Clin Microbiol 27:1631-1635 (1989). cited by applicant .
Stocker, et al., Nanoliter volume, high-resolution NMR
Microspectroscopy using a 60 urn planer microcoil, IEEE Trans.
Biomed. Eng., 44:1122-1127 (1997). cited by applicant .
Miltenyi Biotec, 2007, "autoMACS.TM. Pro Separator",
13/0972-7-91.02, 2007, 8 pages. cited by applicant .
Subramanian, et al., RF Microcoil Design for Practical NMR of
Mass-Limited Samples, J. Magn. Reson., 133:227-231 (1998). cited by
applicant .
Takagi et al., Appl. Environ. Microbiol. 63:4504 (1997). cited by
applicant .
Taktak, et al., Multiparameter Magnetic Relaxation Switch Assays,
Analytical Chemistry, 79(23):8863-8869 (2007). cited by applicant
.
The United States Naval Research Laboratory (NRL), "The FABS
Device: Magnetic Particles", retrieved from
http://www.nrl.navy.mil/chemistry/6170/6177/beads.php on Jan. 8,
2013. cited by applicant .
Torensama, et al., Monoclonal Antibodies Specific for the
Phase-Variant O-Acetylated Ki Capsule of Escerichia coli, J. Clin.
Microbiol., 29(7):1356-1358 (1991). cited by applicant .
Trumbull, et al., Integrating microfabricated fluidic systems and
NMR spectroscopy, IEEE Trans. Biomed. Eng., 47 (1):3-7 (2000).
cited by applicant .
Van Bentum, et al., Towards Nuclear Magnetic Resonance
(MU)-Spectroscopy and (MU)-Imaging, Analyst, Royal Society of
Chemistry, London, 129(9):793-803 (2004). cited by applicant .
Vandeventer, J. Clin. Microbiol. Jul. 2011, 49(7):2533-39. cited by
applicant .
Venkateswaran, et al., Production of Anti-Fibroblast Growth Factor
Receptor Monoclonal Antibodies by In Vitro Immunization, Hybridoma,
11(6):729-739 (1992). cited by applicant .
Verma, Biochim Biophys Acta. 473:1-38 (1977). cited by applicant
.
Vermunt, et al., Isolation of salmonelas by immunomagnetic
separation, J. Appl. Bact., 72:112-118 (1992). cited by applicant
.
Wang et al., 2010, Separation and detection of multiple pathogens
in a food matrix by magnetic SERS nanoprobes, Analytical and
Bioanalytical Chemistry, 399(3): 1271-1278. cited by applicant
.
Wang Hong, Ph.D., "Rapid and Simultaneous Detection of Foodborne
Bacterial Pathogens Using Multiplex Assays", Dissertation Abstract,
University of Arkansas, 2010 (2 Pages). cited by applicant .
Webb and Grant, Signal-to-Noise and Magnetic Susceptibility
Trade-offs in Solenoidal Microcoils for NMR, J. Magn. Reson. B,
113:83-87 (1996). cited by applicant .
Wensink, et al., High Signal to Noise Ratio in Low-field NMR on a
Chip: Simulations and Experimental Results, 17th IEEE MEMS, 407-410
(2004). cited by applicant .
Williams and Wang, Microfabrication of an electromagnetic power
micro-relay using SU-8 based UV-LIGA technology, Microsystem
Technologies, 10(10):699-705 (2004). cited by applicant .
Wu, et al., 1H-NMR Spectroscopy on the Nanoliter Scale for Static
and On-Line Measurements, Anal. Chern., 66:3849 (1994). cited by
applicant .
Yang et al., "Simultaneous Detection of Escherichia coli O157:H7
and Salmonella typhimurium Using Quantum Dots as Fluorescence
Labels", Analyst 131(3), Mar. 2006, pp. 394-101 (8 Pages). cited by
applicant .
Yeung et al., 2002, Quantitative Screening of Yeast
Surface-Displayed Polypeptide Libraries by Magnetic Bead Capture.
Biotechnol. 18:212-220. cited by applicant .
Yu et al. "Development of a Magnetic Microplate
Chemifluorimmunoassay for Rapid Detection of Bacteria and Toxin in
Blood", Analytical Biochemistry 261 (1998), pp. 1-7. cited by
applicant .
Zhao, et al., A rapid bioassay for single bacterial cell
quantitation using bioconjugated nanoparticles, PNAS, 101
(42):15027-15032 (2004). cited by applicant .
Zordan, et al., Detection of Pathogenic E. coli O157:H7 by a Hybrid
Microfluidic SPR and Molecular Imaging Cytometry Device, Cytometry
A, 75A:155-162 (2009). cited by applicant .
Agrawal et al., 1990, Tetrahedron Letters 31:1543-46. cited by
applicant .
Andreassen, Jack, "One micron magnetic beads optimised for
automated immunoassays" as Published in CLI Apr. 2005, retrieved
from
http://www.cli-online.com/uploads/tx_ttproducts/datasheet/one-micron-magn-
etic-beads-optimised-for-automatedimmunoassays.pdf on Dec. 28,
2015, four pages. cited by applicant .
Armenean, et al., Solenoidal and Planar Microcoils for NMR
Spectroscopy, Proc. of the 25th Annual Int. Conf. of the IEEE Eng.
in Med. and Bio. Soc., Cancun, Mexico, Sep. 17, 2003, pp.
3045-3048. cited by applicant .
Barany et al., Gene, 109.2 (1991) "Cloning, overexpression and
nucleotide sequence of a thermostable DNA ligase-encoding gene",
pp. 1-11. cited by applicant .
Barany F. (1991) PNAS 88:189-193. cited by applicant .
Barany, F., Genome research, 1:5-16 (1991) "The Ligase Chain
Reaction in a PCR World". cited by applicant .
Behnia and Webb, Limited-Sample NMR Using Solenoidal Microcoils,
Perfluorocarbon Plugs, and Capillary Spinning, Anal. Chem.,
70:5326-5331 (1998). cited by applicant .
Braslavsky et al., PNAS, 100:3690-3694 (2003). cited by applicant
.
Brown et al., Methods Enzymol., 68:109 (1979). cited by applicant
.
Bruno et al., "Development of an Immunomagnetic Assay System for
Rapid Detection of Bacteria and Leukocytes in Body Fluids," J Mol
Recog, 9 (1996) 474-479. cited by applicant .
Burtis et al. (Burtis, C.A. (Ed.), Tietz Textbook of Clinical
Chemistry, 3rd Edition (1999), W.B. Saunders Company, Philadelphia,
PA, pp. 1793-1794). cited by applicant .
Butter et al., 2002, Synthesis and properties of iron ferrofluids,
J. Magn. Magn. Mater. 252:1-3. cited by applicant .
Byrne, et al., Antibody-Based Sensors: Principles, Problems and
Potential for Detection of Pathogens and Associated Toxins,
Sensors, 9:4407-4445 (2009). cited by applicant .
Campuzano, et al., Bacterial Isolation by Lectin Modified
Microengines, Nano Lett. Jan. 11, 2012; 12(1): 396-401. cited by
applicant .
Cann et al., Proc. Natl. Acad. Sci. 95:14250 (1998) "A
heterodimeric DNA polymerase: Evidence that members of
Euryarchaeota possess a distinct DNA polymerase". cited by
applicant .
Cariello et al., Nucl Acids Res 19:4193 (1991) "Fidelity of
Thermococcus litoralis DNA polymerase (Vent.TM.) in PCR determined
by denaturing gradient gel electrophoresis". cited by applicant
.
Carroll, N. M., E. E. Jaeger, et al. (2000). "Detection of and
discrimination between grampositive and gram-negative bacteria in
intraocular samples by using nested PCR " J Clin 15 Microbiol
38(5): 1753-1757. cited by applicant .
Chandler et al., Automated immunomagnetic separation and microarray
detection of E. coli O157:H7 from poultry carcass rinse, Int. J.
Food Micro., 70 (2001) 143-154. cited by applicant .
Chapman, et al., Use of commercial enzyme immunoassays and
immunomagnetic separation systems for detecting Escherichia coli
O157 in bovine fecal samples, Applied and Environmental
Microbiology, 63(7):2549-2553 (1997). cited by applicant .
Cheng et al., 2012, Concentralion and detection of bacteria in
virtual environmental samples based on non-immunomagnetic
separation and quantum dots by using a laboratory-made system,
Proc. of SPIE:82310Y-1-82310Y-18. cited by applicant .
Chien et al., J. Bacteriol, 127:1550 (1976) "Deoxyribonucleic Acid
Polymerase from the Extreme Thermophile Thermus aquaticus". cited
by applicant .
Wang et al. "Multifunctional Magnetic-OPtical Nanoparticle Probes
for Simultaneous Detection, Separation, and Thermal Ablation of
Multiple Pathogens", Small, vol. 6, No. 2 Jan. 18, 2010, pp.
283-289. cited by applicant .
Ciobanu and Pennington, 3D Micron-scale MRI of Single Biological
Cells, Solid State Nucl. Magn. Reson., 25:138-141 (2004). cited by
applicant .
Cold Spring Harbor Protocols, Recipe for Dulbecco's
phosphate-buffered saline (Dulbecco's PBS, 2009, retrieved from
http://cshprotocols.cshlp.Org/content/2009/3/pdb.red 1725.
full?text_only-true on Mar. 9, 2015, one page. cited by
applicant.
|
Primary Examiner: Gabel; Gailene
Attorney, Agent or Firm: Brown Rudnick LLP Meyers; Thomas
C.
Parent Case Text
RELATED APPLICATION
The present application is a continuation of U.S. non-provisional
patent application Ser. No. 12/850,203, filed Aug. 4, 2010, which
claims the benefit of and priority to U.S. provisional patent
application Ser. No. 61/326,588, filed Apr. 21, 2010, the contents
of each of which are incorporated by reference herein in their
entirety.
Claims
What is claimed is:
1. A method for isolating pathogen in a biological sample, the
method comprising: exposing a body fluid sample comprising blood
cells and pathogen to a plurality of magnetic particles, the
particles comprising at least about 70% magnetic material by weight
and being functionalized for binding pathogen; preventing lysis of
the blood cells in the sample by mixing the particles and the
sample with a buffer that substantially prevents lysis of blood
cells, reduces particle aggregation, and comprises about 75 mM
Tris(hydroxymethyl)-aminomethane hydrochloride, thereby forming
complexes between the pathogen and magnetic particles, said buffer
further comprising about 300 mM NaCl, and about 0.1% polysorbate
20; flowing the complexes through a flow-through capture cell
having a surface upon which a magnetic field is applied; and
isolating complexed pathogen by applying the magnetic field to the
flow-through capture cell, wherein the mixing step is performed
prior to the flowing step.
2. The method of claim 1, wherein the particles are coated with one
or more antibodies capable of binding at least one pathogen.
3. The method of claim 1, wherein the pathogen is selected from the
group consisting of bacteria and viruses.
4. The method of claim 1, wherein the body fluid sample is
blood.
5. A method for isolating pathogen in a biological sample, the
method comprising: diluting a body fluid sample comprising blood
cells and pathogen while preventing lysis of the blood cells by
mixing the sample at a ratio of about 1:1 with a buffer that
substantially prevents lysis of blood cells, reduces particle
aggregation, and comprises about 75 mM
Tris(hydroxymethyl)-aminomethane hydrochloride, about 300 mM NaCl,
and about 0.1% polysorbate 20; exposing the diluted sample to
magnetic nanoparticles, the nanoparticles comprising at least about
70% magnetic material by weight and being functionalized for
binding pathogen; incubating the diluted sample and nanoparticles,
thereby forming complexes between pathogen and the magnetic
nanoparticles; flowing the complexes through a flow-through capture
cell having a surface upon which a magnetic field is applied; and
isolating the complexed pathogen by applying the magnetic field to
the flow-through capture cell, wherein the mixing step is performed
prior to the flowing step.
6. The method of claim 5, wherein the nanoparticles are
functionalized with antibody that specifically binds to
pathogen.
7. The method of claim 6, wherein the pathogen is a bacterium.
8. The method of claim 5, wherein osmolality of the sample is
substantially maintained in the diluting step.
9. The method of claim 5, wherein the incubating step is performed
for from about 10 seconds to about 2 hours.
Description
FIELD OF THE INVENTION
The invention generally relates to using magnetic particles and
magnets to isolate a target analyte from a body fluid sample.
BACKGROUND
Blood-borne pathogens are a significant healthcare problem. A
delayed or improper diagnosis of a bacterial infection can result
in sepsis--a serious, and often deadly, inflammatory response to
the infection. Sepsis is the 10.sup.th leading cause of death in
the United States. Early detection of bacterial infections in blood
is the key to preventing the onset of sepsis. Traditional methods
of detection and identification of blood-borne infection include
blood culture and antibiotic susceptibility assays. Those methods
typically require culturing cells, which can be expensive and can
take as long as 72 hours. Often, septic shock will occur before
cell culture results can be obtained.
Alternative methods for detection of pathogens, particularly
bacteria, have been described by others. Those methods include
molecular detection methods, antigen detection methods, and
metabolite detection methods. Molecular detection methods, whether
involving hybrid capture or polymerase chain reaction (PCR),
require high concentrations of purified DNA for detection. Both
antigen detection and metabolite detection methods also require a
relatively large amount of bacteria and have high limit of
detection (usually>10.sup.4 CFU/mL), thus requiring an
enrichment step prior to detection. This incubation/enrichment
period is intended to allow for the growth of bacteria and an
increase in bacterial cell numbers to more readily aid in
identification. In many cases, a series of two or three separate
incubations is needed to isolate the target bacteria. However, such
enrichment steps require a significant amount of time (e.g., at
least a few days to a week) and can potentially compromise test
sensitivity by killing some of the cells sought to be measured.
There is a need for methods for isolating target analytes, such as
bacteria, from a sample, such as a blood sample, without an
additional enrichment step. There is also a need for methods of
isolating target analytes that are fast and sensitive in order to
provide data for patient treatment decisions in a clinically
relevant time frame.
SUMMARY
The present invention provides methods and devices for isolating
pathogens in a biological sample. The invention allows the rapid
detection of pathogen at very low levels in the sample; thus
enabling early and accurate detection and identification of the
pathogen. The invention is carried out with magnetic particles
having a target-specific binding moiety. Methods of the invention
involve introducing magnetic particles including a target-specific
binding moiety to a body fluid sample in order to create a mixture,
incubating the mixture to allow the particles to bind to a target,
applying a magnetic field to capture target/magnetic particle
complexes on a surface, and washing with a wash solution that
reduces particle aggregation, thereby isolating target/magnetic
particle complexes. A particular advantage of methods of the
invention is for capture and isolation of bacteria and fungi
directly from blood samples at low concentrations that are present
in many clinical samples (as low as 1 CFU/ml of bacteria in a blood
sample).
The target-specific binding moiety will depend on the target to be
captured. The moiety may be any capture moiety known in the art,
such as an antibody, an aptamer, a nucleic acid, a protein, a
receptor, a phage or a ligand. In particular embodiments, the
target-specific binding moiety is an antibody. In certain
embodiments, the antibody is specific for bacteria. In other
embodiments, the antibody is specific for fungi.
The target analyte refers to the target that will be captured and
isolated by methods of the invention. The target may be bacteria,
fungi, protein, a cell, a virus, a nucleic acid, a receptor, a
ligand, or any molecule known in the art. In certain embodiments,
the target is a pathogenic bacteria. In other embodiments, the
target is a gram positive or gram negative bacteria. Exemplary
bacterial species that may be captured and isolated by methods of
the invention include E. coli, Listeria, Clostridium,
Mycobacterium, Shigella, Borrelia, Campylobacter, Bacillus,
Salmonella, Staphylococcus, Enterococcus, Pneumococcus,
Streptococcus, and a combination thereof.
Methods of the invention may be performed with any type of magnetic
particle. Magnetic particles generally fall into two broad
categories. The first category includes particles that are
permanently magnetizable, or ferromagnetic; and the second category
includes particles that demonstrate bulk magnetic behavior only
when subjected to a magnetic field. The latter are referred to as
magnetically responsive particles. Materials displaying
magnetically responsive behavior are sometimes described as
superparamagnetic. However, materials exhibiting bulk ferromagnetic
properties, e.g., magnetic iron oxide, may be characterized as
superparamagnetic when provided in crystals of about 30 nm or less
in diameter. Larger crystals of ferromagnetic materials, by
contrast, retain permanent magnet characteristics after exposure to
a magnetic field and tend to aggregate thereafter due to strong
particle-particle interaction. In certain embodiments, the
particles are superparamagnetic beads. In other embodiments, the
magnetic particles include at least 70% superparamagnetic beads by
weight. In certain embodiments, the superparamagnetic beads are
from about 100 nm to about 250 nm in diameter. In certain
embodiments, the magnetic particle is an iron-containing magnetic
particle. In other embodiments, the magnetic particle includes iron
oxide or iron platinum.
In certain embodiments, the incubating step includes incubating the
mixture in a buffer that inhibits cell lysis. In certain
embodiments, the buffer includes Tris(hydroximethyl)-aminomethane
hydrochloride at a concentration of between about 50 mM and about
100 mM, preferably about 75 mM. In other embodiments, methods of
the invention further include retaining the magnetic particles in a
magnetic field during the washing step. Methods of the invention
may be used with any body fluid. Exemplary body fluids include
blood, sputum, serum, plasma, urine, saliva, sweat, and cerebral
spinal fluid.
Another aspect of the invention provides methods for isolating a
target microorganism from a body fluid sample including introducing
magnetic particles having a target-specific binding moiety to a
body fluid sample in order to create a mixture, incubating the
mixture to allow the particles to bind to the target, applying a
magnetic field to isolate on a surface magnetic particles to which
target is bound, washing the mixture in a wash solution that
reduces particle aggregation, and lysing the captured bacteria and
extracting DNA for further analysis by PCR, microarray
hybridization or sequencing.
Another aspect of the invention provides methods for isolating as
low as 1 CFU/ml of bacteria in a blood sample including introducing
superparamagnetic particles having a diameter from about 100 nm to
about 250 nm and having a bacteria-specific binding moiety to a
body fluid sample in order to create a mixture, incubating said
mixture to allow said particles to bind to a bacteria, applying a
magnetic field to isolate on a surface bacteria/magnetic particle
complexes, and washing the mixture in a wash solution that reduces
particle aggregation, thereby isolating as low as 1 viable CFU/ml
of bacteria in the blood sample.
DETAILED DESCRIPTION
The invention generally relates to using magnetic particles that
capture target pathogens in a body fluid sample and magnets to
isolate the target. Methods of the invention involve introducing
magnetic particles including a target-specific binding moiety to a
body fluid sample in order to create a mixture, incubating the
mixture to allow the particles to bind to a target, applying a
magnetic field to capture target/magnetic particle complexes on a
surface, and washing the mixture in a wash solution that reduces
particle aggregation, thereby isolating target/magnetic particle
complexes. Certain fundamental technologies and principles are
associated with binding magnetic materials to target entities and
subsequently separating by use of magnet fields and gradients. Such
fundamental technologies and principles are known in the art and
have been previously described, such as those described in Janeway
(Immunobiology, 6.sup.th edition, Garland Science Publishing), the
content of which is incorporated by reference herein in its
entirety.
Methods of the invention involve collecting a body fluid having a
target analyte in a container, such as a blood collection tube
(e.g., VACUTAINER, test tube specifically designed for
venipuncture, commercially available from Becton, Dickinson and
company). In certain embodiments, a solution is added that prevents
or reduces aggregation of endogenous aggregating factors, such as
heparin in the case of blood.
A body fluid refers to a liquid material derived from, for example,
a human or other mammal. Such body fluids include, but are not
limited to, mucus, blood, plasma, serum, serum derivatives, bile,
phlegm, saliva, sweat, amniotic fluid, mammary fluid, urine,
sputum, and cerebrospinal fluid (CSF), such as lumbar or
ventricular CSF. A body fluid may also be a fine needle aspirate. A
body fluid also may be media containing cells or biological
material. In particular embodiments, the fluid is blood.
Methods of the invention may be used to detect any target analyte.
The target analyte refers to the substance in the sample that will
be captured and isolated by methods of the invention. The target
may be bacteria, fungi, a protein, a cell (such as a cancer cell, a
white blood cell a virally infected cell, or a fetal cell
circulating in maternal circulation), a virus, a nucleic acid
(e.g., DNA or RNA), a receptor, a ligand, a hormone, a drug, a
chemical substance, or any molecule known in the art. In certain
embodiments, the target is a pathogenic bacteria. In other
embodiments, the target is a gram positive or gram negative
bacteria. Exemplary bacterial species that may be captured and
isolated by methods of the invention include E. coli, Listeria,
Clostridium, Mycobacterium, Shigella, Borrelia, Campylobacter,
Bacillus, Salmonella, Staphylococcus, Enterococcus, Pneumococcus,
Streptococcus, and a combination thereof.
The sample is then mixed with magnetic particles including a
target-specific binding moiety to generate a mixture that is
allowed to incubate such that the particles bind to a target in the
sample, such as a bacterium in a blood sample. The mixture is
allowed to incubate for a sufficient time to allow for the
particles to bind to the target analyte. The process of binding the
magnetic particles to the target analytes associates a magnetic
moment with the target analytes, and thus allows the target
analytes to be manipulated through forces generated by magnetic
fields upon the attached magnetic moment.
In general, incubation time will depend on the desired degree of
binding between the target analyte and the magnetic beads (e.g.,
the amount of moment that would be desirably attached to the
target), the amount of moment per target, the amount of time of
mixing, the type of mixing, the reagents present to promote the
binding and the binding chemistry system that is being employed.
Incubation time can be anywhere from about 5 seconds to a few days.
Exemplary incubation times range from about 10 seconds to about 2
hours. Binding occurs over a wide range of temperatures, generally
between 15.degree. C. and 40.degree. C.
Methods of the invention may be performed with any type of magnetic
particle. Production of magnetic particles and particles for use
with the invention are known in the art. See for example Giaever
(U.S. Pat. No. 3,970,518), Senyi et al. (U.S. Pat. No. 4,230,685),
Dodin et al. (U.S. Pat. No. 4,677,055), Whitehead et al. (U.S. Pat.
No. 4,695,393), Benjamin et al. (U.S. Pat. No. 5,695,946), Giaever
(U.S. Pat. No. 4,018,886), Rembaum (U.S. Pat. No. 4,267,234),
Molday (U.S. Pat. No. 4,452,773), Whitehead et al. (U.S. Pat. No.
4,554,088), Forrest (U.S. Pat. No. 4,659,678), Liberti et al. (U.S.
Pat. No. 5,186,827), Own et al. (U.S. Pat. No. 4,795,698), and
Liberti et al. (WO 91/02811), the content of each of which is
incorporated by reference herein in its entirety.
Magnetic particles generally fall into two broad categories. The
first category includes particles that are permanently
magnetizable, or ferromagnetic; and the second category includes
particles that demonstrate bulk magnetic behavior only when
subjected to a magnetic field. The latter are referred to as
magnetically responsive particles. Materials displaying
magnetically responsive behavior are sometimes described as
superparamagnetic. However, materials exhibiting bulk ferromagnetic
properties, e.g., magnetic iron oxide, may be characterized as
superparamagnetic when provided in crystals of about 30 nm or less
in diameter. Larger crystals of ferromagnetic materials, by
contrast, retain permanent magnet characteristics after exposure to
a magnetic field and tend to aggregate thereafter due to strong
particle-particle interaction. In certain embodiments, the
particles are superparamagnetic beads. In certain embodiments, the
magnetic particle is an iron containing magnetic particle. In other
embodiments, the magnetic particle includes iron oxide or iron
platinum.
In certain embodiments, the magnetic particles include at least
about 10% superparamagnetic beads by weight, at least about 20%
superparamagnetic beads by weight, at least about 30%
superparamagnetic beads by weight, at least about 40%
superparamagnetic beads by weight, at least about 50%
superparamagnetic beads by weight, at least about 60%
superparamagnetic beads by weight, at least about 70%
superparamagnetic beads by weight, at least about 80%
superparamagnetic beads by weight, at least about 90%
superparamagnetic beads by weight, at least about 95%
superparamagnetic beads by weight, or at least about 99%
superparamagnetic beads by weight. In a particular embodiment, the
magnetic particles include at least about 70% superparamagnetic
beads by weight.
In certain embodiments, the superparamagnetic beads are less than
100 nm in diameter. In other embodiments, the superparamagnetic
beads are about 150 nm in diameter, are about 200 nm in diameter,
are about 250 nm in diameter, are about 300 nm in diameter, are
about 350 nm in diameter, are about 400 nm in diameter, are about
500 nm in diameter, or are about 1000 nm in diameter. In a
particular embodiment, the superparamagnetic beads are from about
100 nm to about 250 nm in diameter.
In certain embodiments, the particles are beads (e.g.,
nanoparticles) that incorporate magnetic materials, or magnetic
materials that have been functionalized, or other configurations as
are known in the art. In certain embodiments, nanoparticles may be
used that include a polymer material that incorporates magnetic
material(s), such as nanometal material(s). When those nanometal
material(s) or crystal(s), such as Fe.sub.3O.sub.4, are
superparamagnetic, they may provide advantageous properties, such
as being capable of being magnetized by an external magnetic field,
and demagnetized when the external magnetic field has been removed.
This may be advantageous for facilitating sample transport into and
away from an area where the sample is being processed without undue
bead aggregation.
One or more or many different nanometal(s) may be employed, such as
Fe.sub.3O.sub.4, FePt, or Fe, in a core-shell configuration to
provide stability, and/or various others as may be known in the
art. In many applications, it may be advantageous to have a
nanometal having as high a saturated moment per volume as possible,
as this may maximize gradient related forces, and/or may enhance a
signal associated with the presence of the beads. It may also be
advantageous to have the volumetric loading in a bead be as high as
possible, for the same or similar reason(s). In order to maximize
the moment provided by a magnetizable nanometal, a certain
saturation field may be provided. For example, for Fe.sub.3O.sub.4
superparamagnetic particles, this field may be on the order of
about 0.3 T.
The size of the nanometal containing bead may be optimized for a
particular application, for example, maximizing moment loaded upon
a target, maximizing the number of beads on a target with an
acceptable detectability, maximizing desired force-induced motion,
and/or maximizing the difference in attached moment between the
labeled target and non-specifically bound targets or bead
aggregates or individual beads. While maximizing is referenced by
example above, other optimizations or alterations are contemplated,
such as minimizing or otherwise desirably affecting conditions.
In an exemplary embodiment, a polymer bead containing 80 wt %
Fe.sub.3O.sub.4 superparamagnetic particles, or for example, 90 wt
% or higher superparamagnetic particles, is produced by
encapsulating superparamagnetic particles with a polymer coating to
produce a bead having a diameter of about 250 nm.
Magnetic particles for use with methods of the invention have a
target-specific binding moiety that allows for the particles to
specifically bind the target of interest in the sample. The
target-specific moiety may be any molecule known in the art and
will depend on the target to be captured and isolated. Exemplary
target-specific binding moieties include nucleic acids, proteins,
ligands, antibodies, aptamers, and receptors.
In particular embodiments, the target-specific binding moiety is an
antibody, such as an antibody that binds a particular bacterium.
General methodologies for antibody production, including criteria
to be considered when choosing an animal for the production of
antisera, are described in Harlow et al. (Antibodies, Cold Spring
Harbor Laboratory, pp. 93-117, 1988). For example, an animal of
suitable size such as goats, dogs, sheep, mice, or camels are
immunized by administration of an amount of immunogen, such the
target bacteria, effective to produce an immune response. An
exemplary protocol is as follows. The animal is injected with 100
milligrams of antigen resuspended in adjuvant, for example Freund's
complete adjuvant, dependent on the size of the animal, followed
three weeks later with a subcutaneous injection of 100 micrograms
to 100 milligrams of immunogen with adjuvant dependent on the size
of the animal, for example Freund's incomplete adjuvant. Additional
subcutaneous or intraperitoneal injections every two weeks with
adjuvant, for example Freund's incomplete adjuvant, are
administered until a suitable titer of antibody in the animal's
blood is achieved. Exemplary titers include a titer of at least
about 1:5000 or a titer of 1:100,000 or more, i.e., the dilution
having a detectable activity. The antibodies are purified, for
example, by affinity purification on columns containing protein G
resin or target-specific affinity resin.
The technique of in vitro immunization of human lymphocytes is used
to generate monoclonal antibodies. Techniques for in vitro
immunization of human lymphocytes are well known to those skilled
in the art. See, e.g., Inai, et al., Histochemistry, 99(5):335 362,
May 1993; Mulder, et al., Hum. Immunol., 36(3):186 192, 1993;
Harada, et al., J. Oral Pathol. Med., 22(4):145 152, 1993; Stauber,
et al., J. Immunol. Methods, 161(2):157 168, 1993; and
Venkateswaran, et al., Hybridoma, 11(6) 729 739, 1992. These
techniques can be used to produce antigen-reactive monoclonal
antibodies, including antigen-specific IgG, and IgM monoclonal
antibodies.
Any antibody or fragment thereof having affinity and specific for
the bacteria of interest is within the scope of the invention
provided herein. Immunomagnetic beads against Salmonella are
provided in Vermunt et al. (J. Appl. Bact. 72:112, 1992).
Immunomagnetic beads against Staphylococcus aureus are provided in
Johne et al. (J. Clin. Microbiol. 27:1631, 1989). Immunomagnetic
beads against Listeria are provided in Skjerve et al. (Appl. Env.
Microbiol. 56:3478, 1990). Immunomagnetic beads against Escherichia
coli are provided in Lund et al. (J. Clin. Microbiol. 29:2259,
1991).
Methods for attaching the target-specific binding moiety to the
magnetic particle are known in the art. Coating magnetic particles
with antibodies is well known in the art, see for example Harlow et
al. (Antibodies, Cold Spring Harbor Laboratory, 1988), Hunter et
al. (Immunoassays for Clinical Chemistry, pp. 147-162, eds.,
Churchill Livingston, Edinborough, 1983), and Stanley (Essentials
in Immunology and Serology, Delmar, pp. 152-153, 2002). Such
methodology can easily be modified by one of skill in the art to
bind other types of target-specific binding moieties to the
magnetic particles. Certain types of magnetic particles coated with
a functional moiety are commercially available from Sigma-Aldrich
(St. Louis, Mo.).
In certain embodiments, a buffer solution is added to the sample
along with the magnetic beads. An exemplary buffer includes
Tris(hydroximethyl)-aminomethane hydrochloride at a concentration
of about 75 mM. It has been found that the buffer composition,
mixing parameters (speed, type of mixing, such as rotation, shaking
etc., and temperature) influence binding. It is important to
maintain osmolality of the final solution (e.g., blood+buffer) to
maintain high label efficiency. In certain embodiments, buffers
used in methods of the invention are designed to prevent lysis of
blood cells, facilitate efficient binding of targets with magnetic
beads and to reduce formation of bead aggregates. It has been found
that the buffer solution containing 300 mM NaCl, 75 mM Tris-HCl pH
8.0 and 0.1% polysorbate 20, which is sold under the trade name
Tween 20 by MilliporeSigma (St. Louis, Mo.), meets these design
goals.
Without being limited by any particular theory or mechanism of
action, it is believed that sodium chloride is mainly responsible
for maintaining osmolality of the solution and for the reduction of
non-specific binding of magnetic bead through ionic interaction.
Tris(hydroximethyl)-aminomethane hydrochloride is a well
established buffer compound frequently used in biology to maintain
pH of a solution. It has been found that 75 mM concentration is
beneficial and sufficient for high binding efficiency. Likewise,
polysorbate 20 is widely used as a mild detergent to decrease
nonspecific attachment due to hydrophobic interactions. Various
assays use polysorbate 20 at concentrations ranging from 0.01% to
1%. The 0.1% concentration appears to be optimal for the efficient
labeling of bacteria, while maintaining blood cells intact
An alternative approach to achieve high binding efficiency while
reducing time required for the binding step is to use static mixer,
or other mixing devices that provide efficient mixing of viscous
samples at high flow rates, such as at or around 5 mL/min. In one
embodiment, the sample is mixed with binding buffer in ratio of, or
about, 1:1, using a mixing interface connector. The diluted sample
then flows through a mixing interface connector where it is mixed
with target-specific nanoparticles. Additional mixing interface
connectors providing mixing of sample and antigen-specific
nanoparticles can be attached downstream to improve binding
efficiency. The combined flow rate of the labeled sample is
selected such that it is compatible with downstream processing.
After binding of the magnetic particles to the target analyte in
the mixture to form target/magnetic particle complexes, a magnetic
field is applied to the mixture to capture the complexes on a
surface. Components of the mixture that are not bound to magnetic
particles will not be affected by the magnetic field and will
remain free in the mixture. Methods and apparatuses for separating
target/magnetic particle complexes from other components of a
mixture are known in the art. For example, a steel mesh may be
coupled to a magnet, a linear channel or channels may be configured
with adjacent magnets, or quadrapole magnets with annular flow may
be used. Other methods and apparatuses for separating
target/magnetic particle complexes from other components of a
mixture are shown in Rao et al. (U.S. Pat. No. 6,551,843), Liberti
et al. (U.S. Pat. No. 5,622,831), Hatch et al. (U.S. Pat. No.
6,514,415), Benjamin et al. (U.S. Pat. No. 5,695,946), Liberti et
al. (U.S. Pat. No. 5,186,827), Wang et al. (U.S. Pat. No.
5,541,072), Liberti et al. (U.S. Pat. No. 5,466,574), and
Terstappen et al. (U.S. Pat. No. 6,623,983), the content of each of
which is incorporated by reference herein in its entirety.
In certain embodiments, the magnetic capture is achieved at high
efficiency by utilizing a flow-through capture cell with a number
of strong rare earth bar magnets placed perpendicular to the flow
of the sample. When using a flow chamber with flow path
cross-section 0.5 mm.times.20 mm (h.times.w) and 7 bar NdFeB
magnets, the flow rate could be as high as 5 mL/min or more, while
achieving capture efficiency close to 100%.
The above described type of magnetic separation produces efficient
capture of a target analyte and the removal of a majority of the
remaining components of a sample mixture. However, such a process
produces a sample that contains a very high percent of magnetic
particles that are not bound to target analytes because the
magnetic particles are typically added in excess, as well as
non-specific target entities. Non-specific target entities may for
example be bound at a much lower efficiency, for example 1% of the
surface area, while a target of interest might be loaded at 50% or
nearly 100% of the available surface area or available antigenic
cites. However, even 1% loading may be sufficient to impart force
necessary for trapping in a magnetic gradient flow cell or sample
chamber.
For example, in the case of immunomagnetic binding of bacteria or
fungi in a blood sample, the sample may include: bound targets at a
concentration of about 1/mL or a concentration less than about
10.sup.6/mL; background particles at a concentration of about
10.sup.7/ml to about 10.sup.10/ml; and non-specific targets at a
concentration of about 10/ml to about 10.sup.5/ml.
The presence of magnetic particles that are not bound to target
analytes and non-specific target entities on the surface that
includes the target/magnetic particle complexes interferes with the
ability to successfully detect the target of interest. The magnetic
capture of the resulting mix, and close contact of magnetic
particles with each other and bound targets, result in the
formation of aggregate that is hard to dispense and which might be
resistant or inadequate for subsequent processing or analysis
steps. In order to remove magnetic particles that are not bound to
target analytes and non-specific target entities, methods of the
invention involve washing the surface with a wash solution that
reduces particle aggregation, thereby isolating target/magnetic
particle complexes from the magnetic particles that are not bound
to target analytes and non-specific target entities. The wash
solution minimizes the formation of the aggregates.
Methods of the invention may use any wash solution that imparts a
net negative charge to the magnetic particle that is not sufficient
to disrupt interaction between the target-specific moiety of the
magnetic particle and the target analyte. Without being limited by
any particular theory or mechanism of action, it is believed that
attachment of the negatively charged molecules in the wash solution
to magnetic particles provides net negative charge to the particles
and facilitates dispersal of non-specifically aggregated particles.
At the same time, the net negative charge is not sufficient to
disrupt strong interaction between the target-specific moiety of
the magnetic particle and the target analyte (e.g., an
antibody-antigen interaction). Exemplary solutions include heparin,
Tris-HCl, Tris-borate-EDTA (TBE), Tris-acetate-EDTA (TAE),
Tris-cacodylate, HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid), PBS
(phosphate buffered saline), PIPES
(piperazine-N,N'-bis(2-ethanesulfonic acid), MES
(2-N-morpholino)ethanesulfonic acid), Tricine
(N-(Tri(hydroximethyl)methyl)glycine), and similar buffering
agents. In certain embodiments, only a single wash cycle is
performed. In other embodiments, more than one wash cycle is
performed.
In particular embodiments, the wash solution includes heparin. For
embodiments in which the body fluid sample is blood, the heparin
also reduces probability of clotting of blood components after
magnetic capture. The bound targets are washed with
heparin-containing buffer 1-3 times to remove blood components and
to reduce formation of aggregates.
Once the target/magnetic particle complexes are isolated, the
target may be analyzed by a multitude of existing technologies,
such as miniature NMR , Polymerase Chain Reaction (PCR), mass
spectrometry, fluorescent labeling and visualization using
microscopic observation, fluorescent in situ hybridization (FISH),
growth-based antibiotic sensitivity tests, and variety of other
methods that may be conducted with purified target without
significant contamination from other sample components. In one
embodiment, isolated bacteria are lysed with a chaotropic solution,
and DNA is bound to DNA extraction resin. After washing of the
resin, the bacterial DNA is eluted and used in quantitative RT-PCR
to detect the presence of a specific species, and/or, subclasses of
bacteria.
In another embodiment, captured bacteria is removed from the
magnetic particles to which they are bound and the processed sample
is mixed with fluorescent labeled antibodies specific to the
bacteria or fluorescent Gram stain. After incubation, the reaction
mixture is filtered through 0.2 .mu.m to 1.0 .mu.m filter to
capture labeled bacteria while allowing majority of free beads and
fluorescent labels to pass through the filter. Bacteria is
visualized on the filter using microscopic techniques, e.g. direct
microscopic observation, laser scanning or other automated methods
of image capture. The presence of bacteria is detected through
image analysis. After the positive detection by visual techniques,
the bacteria can be further characterized using PCR or genomic
methods.
Detection of bacteria of interest can be performed by use of
nucleic acid probes following procedures which are known in the
art. Suitable procedures for detection of bacteria using nucleic
acid probes are described, for example, in Stackebrandt et al.
(U.S. Pat. No. 5,089,386), King et al. (WO 90/08841), Foster et al.
(WO 92/15883), and Cossart et al. (WO 89/06699), each of which is
hereby incorporated by reference.
A suitable nucleic acid probe assay generally includes sample
treatment and lysis, hybridization with selected probe(s), hybrid
capture, and detection. Lysis of the bacteria is necessary to
release the nucleic acid for the probes. The nucleic acid target
molecules are released by treatment with any of a number of lysis
agents, including alkali (such as NaOH), guanidine salts (such as
guanidine thiocyanate), enzymes (such as lysozyme, mutanolysin and
proteinase K), and detergents. Lysis of the bacteria, therefore,
releases both DNA and RNA, particularly ribosomal RNA and
chromosomal DNA both of which can be utilized as the target
molecules with appropriate selection of a suitable probe. Use of
rRNA as the target molecule(s), may be advantageous because rRNAs
constitute a significant component of cellular mass, thereby
providing an abundance of target molecules. The use of rRNA probes
also enhances specificity for the bacteria of interest, that is,
positive detection without undesirable cross-reactivity which can
lead to false positives or false detection.
Hybridization includes addition of the specific nucleic acid
probes. In general, hybridization is the procedure by which two
partially or completely complementary nucleic acids are combined,
under defined reaction conditions, in an anti-parallel fashion to
form specific and stable hydrogen bonds. The selection or
stringency of the hybridization/reaction conditions is defined by
the length and base composition of the probe/target duplex, as well
as by the level and geometry of mis-pairing between the two nucleic
acid strands. Stringency is also governed by such reaction
parameters as temperature, types and concentrations of denaturing
agents present and the type and concentration of ionic species
present in the hybridization solution.
The hybridization phase of the nucleic acid probe assay is
performed with a single selected probe or with a combination of
two, three or more probes. Probes are selected having sequences
which are homologous to unique nucleic acid sequences of the target
organism. In general, a first capture probe is utilized to capture
formed hybrid molecules. The hybrid molecule is then detected by
use of antibody reaction or by use of a second detector probe which
may be labelled with a radioisotope (such as phosphorus-32) or a
fluorescent label (such as fluorescein) or chemiluminescent
label.
Detection of bacteria of interest can also be performed by use of
PCR techniques. A suitable PCR technique is described, for example,
in Verhoef et al. (WO 92/08805). Such protocols may be applied
directly to the bacteria captured on the magnetic beads. The
bacteria is combined with a lysis buffer and collected nucleic acid
target molecules are then utilized as the template for the PCR
reaction.
For detection of the selected bacteria by use of antibodies,
isolated bacteria are contacted with antibodies specific to the
bacteria of interest. As noted above, either polyclonal or
monoclonal antibodies can be utilized, but in either case have
affinity for the particular bacteria to be detected. These
antibodies, will adhere/bind to material from the specific target
bacteria. With respect to labeling of the antibodies, these are
labeled either directly or indirectly with labels used in other
known immunoassays. Direct labels may include fluorescent,
chemiluminescent, bioluminescent, radioactive, metallic, biotin or
enzymatic molecules. Methods of combining these labels to
antibodies or other macromolecules are well known to those in the
art. Examples include the methods of Hijmans, W. et al. (1969),
Clin. Exp. Immunol. 4, 457-, for fluorescein isothiocyanate, the
method of Goding, J. W. (1976), J. Immunol. Meth. 13, 215-, for
tetramethylrhodamine isothiocyanate, and the method of Ingrall, E.
(1980), Meth. in Enzymol. 70, 419-439 for enzymes.
These detector antibodies may also be labeled indirectly. In this
case the actual detection molecule is attached to a secondary
antibody or other molecule with binding affinity for the
anti-bacteria cell surface antibody. If a secondary antibody is
used it is preferably a general antibody to a class of antibody
(IgG and IgM) from the animal species used to raise the
anti-bacteria cell surface antibodies. For example, the second
antibody may be conjugated to an enzyme, either alkaline
phosphatase or to peroxidase. To detect the label, after the
bacteria of interest is contacted with the second antibody and
washed, the isolated component of the sample is immersed in a
solution containing a chromogenic substrate for either alkaline
phosphatase or peroxidase. A chromogenic substrate is a compound
that can be cleaved by an enzyme to result in the production of
some type of detectable signal which only appears when the
substrate is cleaved from the base molecule. The chromogenic
substrate is colorless, until it reacts with the enzyme, at which
time an intensely colored product is made. Thus, material from the
bacteria colonies adhered to the membrane sheet will become an
intense blue/purple/black color, or brown/red while material from
other colonies will remain colorless. Examples of detection
molecules include fluorescent substances, such as
4-methylumbelliferyl phosphate, and chromogenic substances, such as
4-nitrophenylphosphate, 3,3',5,5'-tetramethylbenzidine and
2,2'-azino-di-[3-ethelbenz-thiazoliane sulfonate (6)]. In addition
to alkaline phosphatase and peroxidase, other useful enzymes
include .quadrature.-galactosidase, .quadrature.-glucuronidase,
.quadrature.-glucosidase, .quadrature.-glucosidase,
.quadrature.-mannosidase, galactose oxidase, glucose oxidase and
hexokinase.
Detection of bacteria of interest using NMR may be accomplished as
follows. In the use of NMR as a detection methodology, in which a
sample is delivered to a detector coil centered in a magnet, the
target of interest, such as a magnetically labeled bacterium, may
be delivered by a fluid medium, such as a fluid substantially
composed of water. In such a case, the magnetically labeled target
may go from a region of very low magnetic field to a region of high
magnetic field, for example, a field produced by an about 1 to
about 2 Tesla magnet. In this manner, the sample may traverse a
magnetic gradient, on the way into the magnet and on the way out of
the magnet. As may be seen via equations 1 and 2 below, the target
may experience a force pulling into the magnet in the direction of
sample flow on the way into the magnet, and a force into the magnet
in the opposite direction of flow on the way out of the magnet. The
target may experience a retaining force trapping the target in the
magnet if flow is not sufficient to overcome the gradient force. m
dot (del B)=F Equation 1 v.sub.t=-F/(6*p*n*r) Equation 2 where n is
the viscosity, r is the bead diameter, F is the vector force, B is
the vector field, and m is the vector moment of the bead
Magnetic fields on a path into a magnet may be non-uniform in the
transverse direction with respect to the flow into the magnet. As
such, there may be a transverse force that pulls targets to the
side of a container or a conduit that provides the sample flow into
the magnet. Generally, the time it takes a target to reach the wall
of a conduit is associated with the terminal velocity and is lower
with increasing viscosity. The terminal velocity is associated with
the drag force, which may be indicative of creep flow in certain
cases. In general, it may be advantageous to have a high viscosity
to provide a higher drag force such that a target will tend to be
carried with the fluid flow through the magnet without being
trapped in the magnet or against the conduit walls.
Newtonian fluids have a flow characteristic in a conduit, such as a
round pipe, for example, that is parabolic, such that the flow
velocity is zero at the wall, and maximal at the center, and having
a parabolic characteristic with radius. The velocity decreases in a
direction toward the walls, and it is easier to magnetically trap
targets near the walls, either with transverse gradients force on
the target toward the conduit wall, or in longitudinal gradients
sufficient to prevent target flow in the pipe at any position. In
order to provide favorable fluid drag force to keep the samples
from being trapped in the conduit, it may be advantageous to have a
plug flow condition, wherein the fluid velocity is substantially
uniform as a function of radial position in the conduit.
When NMR detection is employed in connection with a flowing sample,
the detection may be based on a perturbation of the NMR water
signal caused by a magnetically labeled target (Sillerud et al.,
JMR (Journal of Magnetic Resonance), vol. 181, 2006). In such a
case, the sample may be excited at time 0, and after some delay,
such as about 50 ms or about 100 ms, an acceptable measurement
(based on a detected NMR signal) may be produced. Alternatively,
such a measurement may be produced immediately after excitation,
with the detection continuing for some duration, such as about 50
ms or about 100 ms. It may be advantageous to detect the NMR signal
for substantially longer time durations after the excitation.
By way of example, the detection of the NMR signal may continue for
a period of about 2 seconds in order to record spectral information
at high-resolution. In the case of parabolic or Newtonian flow, the
perturbation excited at time 0 is typically smeared because the
water around the perturbation source travels at different velocity,
depending on radial position in the conduit. In addition, spectral
information may be lost due to the smearing or mixing effects of
the differential motion of the sample fluid during signal
detection. When carrying out an NMR detection application involving
a flowing fluid sample, it may be advantageous to provide plug-like
sample flow to facilitate desirable NMR contrast and/or desirable
NMR signal detection.
Differential motion within a flowing Newtonian fluid may have
deleterious effects in certain situations, such as a situation in
which spatially localized NMR detection is desired, as in magnetic
resonance imaging. In one example, a magnetic object, such as a
magnetically labeled bacterium, is flowed through the NMR detector
and its presence and location are detected using MRI techniques.
The detection may be possible due to the magnetic field of the
magnetic object, since this field perturbs the magnetic field of
the fluid in the vicinity of the magnetic object. The detection of
the magnetic object is improved if the fluid near the object
remains near the object. Under these conditions, the magnetic
perturbation may be allowed to act longer on any given volume
element of the fluid, and the volume elements of the fluid so
affected will remain in close spatial proximity. Such a stronger,
more localized magnetic perturbation will be more readily detected
using NMR or MRI techniques.
If a Newtonian fluid is used to carry the magnetic objects through
the detector, the velocity of the fluid volume elements will depend
on radial position in the fluid conduit. In such a case, the fluid
near a magnetic object will not remain near the magnetic object as
the object flows through the detector. The effect of the magnetic
perturbation of the object on the surrounding fluid may be smeared
out in space, and the strength of the perturbation on any one fluid
volume element may be reduced because that element does not stay
within range of the perturbation. The weaker, less-well-localized
perturbation in the sample fluid may be undetectable using NMR or
MRI techniques.
Certain liquids, or mixtures of liquids, exhibit non-parabolic flow
profiles in circular conduits. Such fluids may exhibit
non-Newtonian flow profiles in other conduit shapes. The use of
such a fluid may prove advantageous as the detection fluid in an
application employing an NMR-based detection device. Any such
advantageous effect may be attributable to high viscosity of the
fluid, a plug-like flow profile associated with the fluid, and/or
other characteristic(s) attributed to the fluid that facilitate
detection. As an example, a shear-thinning fluid of high viscosity
may exhibit a flow velocity profile that is substantially uniform
across the central regions of the conduit cross-section. The
velocity profile of such a fluid may transition to a zero or very
low value near or at the walls of the conduit, and this transition
region may be confined to a very thin layer near the wall.
Not all fluids, or all fluid mixtures, are compatible with the NMR
detection methodology. In one example, a mixture of glycerol and
water can provide high viscosity, but the NMR measurement is
degraded because separate NMR signals are detected from the water
and glycerol molecules making up the mixture. This can undermine
the sensitivity of the NMR detector. In another example, the
non-water component of the fluid mixture can be chosen to have no
NMR signal, which may be achieved by using a perdeuterated fluid
component, for example, or using a perfluorinated fluid component.
This approach may suffer from the loss of signal intensity since a
portion of the fluid in the detection coil does not produce a
signal.
Another approach may be to use a secondary fluid component that
constitutes only a small fraction of the total fluid mixture. Such
a low-concentration secondary fluid component can produce an NMR
signal that is of negligible intensity when compared to the signal
from the main component of the fluid, which may be water. It may be
advantageous to use a low-concentration secondary fluid component
that does not produce an NMR signal in the detector. For example, a
perfluorinated or perdeuterated secondary fluid component may be
used. The fluid mixture used in the NMR detector may include one,
two, or more than two secondary components in addition to the main
fluid component. The fluid components employed may act in concert
to produce the desired fluid flow characteristics, such as
high-viscosity and/or plug flow. The fluid components may be useful
for providing fluid characteristics that are advantageous for the
performance of the NMR detector, for example by providing NMR
relaxation times that allow faster operation or higher signal
intensities.
A non-Newtonian fluid may provide additional advantages for the
detection of objects by NMR or MRI techniques. As one example, the
objects being detected may all have substantially the same velocity
as they go through the detection coil. This characteristic velocity
may allow simpler or more robust algorithms for the analysis of the
detection data. As another example, the objects being detected may
have fixed, known, and uniform velocity. This may prove
advantageous in devices where the position of the detected object
at later times is needed, such as in a device that has a
sequestration chamber or secondary detection chamber down-stream
from the NMR or MRI detection coil, for example.
In an exemplary embodiment, sample delivery into and out of a 1.7 T
cylindrical magnet using a fluid delivery medium containing 0.1% to
0.5% Xanthan gum in water was successfully achieved. Such delivery
is suitable to provide substantially plug-like flow, high
viscosity, such as from about 10 cP to about 3000 cP, and good NMR
contrast in relation to water. Xanthan gum acts as a non-Newtonian
fluid, having characteristics of a non-Newtonian fluid that are
well know in the art, and does not compromise NMR signal
characteristics desirable for good detection in a desirable mode of
operation.
In certain embodiments, methods of the invention are useful for
direct detection of bacteria from blood. Such a process is
described here. Sample is collected in sodium heparin tube by
venipuncture, acceptable sample volume is about 1 mL to 10 mL.
Sample is diluted with binding buffer and superparamagnetic
particles having target-specific binding moieties are added to the
sample, followed by incubation on a shaking incubator at 37.degree.
C. for about 30 min to 120 min. Alternative mixing methods can also
be used. In a particular embodiment, sample is pumped through a
static mixer, such that reaction buffer and magnetic beads are
added to the sample as the sample is pumped through the mixer. This
process allows for efficient integration of all components into a
single fluidic part, avoids moving parts and separate incubation
vessels and reduces incubation time.
Capture of the labeled targets allows for the removal of blood
components and reduction of sample volume from 30 mL to 5 mL. The
capture is performed in a variety of magnet/flow configurations. In
certain embodiments, methods include capture in a sample tube on a
shaking platform or capture in a flow-through device at flow rate
of 5 mL/min, resulting in total capture time of 6 min.
After capture, the sample is washed with wash buffer including
heparin to remove blood components and free beads. The composition
of the wash buffer is optimized to reduce aggregation of free
beads, while maintaining the integrity of the bead/target
complexes.
The detection method is based on a miniature NMR detector tuned to
the magnetic resonance of water. When the sample is magnetically
homogenous (no bound targets), the NMR signal from water is clearly
detectable and strong. The presence of magnetic material in the
detector coil disturbs the magnetic field, resulting in reduction
in water signal. One of the primary benefits of this detection
method is that there is no magnetic background in biological
samples which significantly reduces the requirements for stringency
of sample processing. In addition, since the detected signal is
generated by water, there is a built-in signal amplification which
allows for the detection of a single labeled bacterium.
This method provides for isolation and detection of as low as or
even lower than 1 CFU/ml of bacteria in a blood sample.
Methods of the invention may also be combined with other separation
and isolation protocols known in the art. Particularly, methods of
the invention may be combined with methods shown in co-pending and
co-owned U.S. Pat. No. 9,389,225, entitled Separating Target
Analytes Using Alternating Magnetic Fields, the content of which is
incorporated by reference herein in its entirety.
INCORPORATION BY REFERENCE
References and citations to other documents, such as patents,
patent applications, patent publications, journals, books, papers,
web contents, have been made throughout this disclosure. All such
documents are hereby incorporated herein by reference in their
entirety for all purposes.
EQUIVALENTS
Various modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including references to the scientific and patent
literature cited herein. The subject matter herein contains
important information, exemplification and guidance that can be
adapted to the practice of this invention in its various
embodiments and equivalents thereof.
EXAMPLES
Example 1: Sample
Blood samples from healthy volunteers were spiked with clinically
relevant concentrations of bacteria (1-10 CFU/mL) including both
laboratory strains and clinical isolates of the bacterial species
most frequently found in bloodstream infections.
Example 2 Antibody Preparation
In order to generate polyclonal, pan-Gram-positive
bacteria-specific IgG, a goat was immunized by first administering
bacterial antigens suspended in complete Freund's adjuvant intra
lymph node, followed by subcutaneous injection of bacterial
antigens in incomplete Freund's adjuvant in 2 week intervals. The
antigens were prepared for antibody production by growing bacteria
to exponential phase (OD.sub.600=0.4-0.8). Following harvest of the
bacteria by centrifugation, the bacteria was inactivated using
formalin fixation in 4% formaldehyde for 4 hr at 37.degree. C.
After 3 washes of bacteria with PBS (15 min wash, centrifugation
for 20 min at 4000 rpm) the antigen concentration was measured
using BCA assay and the antigen was used at 1 mg/mL for
immunization. In order to generate Gram-positive bacteria-specific
IgG, several bacterial species were used for inoculation:
Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus
faecium and Enterococcus fecalis.
The immune serum was purified using affinity chromatography on a
protein G sepharose column (GE Healthcare), and reactivity was
determined using ELISA. Antibodies cross-reacting with
Gram-negative bacteria and fungi were removed by absorption of
purified IgG with formalin-fixed Gram-negative bacteria and fungi.
The formalin-fixed organisms were prepared similar to as described
above and mixed with IgG. After incubation for 2 hrs at room
temperature, the preparation was centrifuged to remove bacteria.
Final antibody preparation was clarified by centrifugation and used
for the preparation of antigen-specific magnetic beads.
Example 3: Preparation of Antigen-Specific Magnetic Beads
Superparamagnetic beads were synthesized by encapsulating iron
oxide nanoparticles (5-15 nm diameter) in a latex core and labeling
with goat IgG. Ferrofluid containing nanoparticles in organic
solvent was precipitated with ethanol, nanoparticles were
resuspended in aqueous solution of styrene and surfactant Hitenol
BC-10, and emulsified using sonication. The mixture was allowed to
equilibrate overnight with stirring and filtered through 1.2 and
0.45 .mu.m filters to achieve uniform micelle size. Styrene,
acrylic acid and divynilbenzene were added in carbonate buffer at
pH 9.6. The polymerization was initiated in a mixture at 70.degree.
C. with the addition of K.sub.2S.sub.2O.sub.8 and the reaction was
allowed to complete overnight. The synthesized particles were
washed 3 times with 0.1% SDS using magnetic capture, filtered
through 1.2, 0.8, and 0.45 .mu.m filters and used for antibody
conjugation.
The production of beads resulted in a distribution of sizes that
may be characterized by an average size and a standard deviation.
In the case of labeling and extracting of bacteria from blood, the
average size for optimal performance was found to be between 100
and 350 nm, for example between 200 nm to 250 nm.
The purified IgG were conjugated to prepared beads using standard
chemistry. After conjugation, the beads were resuspended in 0.1%
BSA which is used to block non-specific binding sites on the bead
and to increase the stability of bead preparation.
Example 4: Labeling of Rare Cells Using Excess of Magnetic
Nanoparticles
Bacteria, present in blood during blood-stream infection, were
magnetically labeled using the superparamagnetic beads prepared in
Example 3 above. The spiked samples as described in Example 1 were
diluted 3-fold with a Tris-based binding buffer and target-specific
beads, followed by incubation on a shaking platform at 37.degree.
C. for up to 2 hr. After incubation, the labeled targets were
magnetically separated followed by a wash step designed to remove
blood products. See example 5 below.
Example 5: Magnetic Capture of Bound Bacteria
Blood including the magnetically labeled target bacteria and excess
free beads were injected into a flow-through capture cell with a
number of strong rare earth bar magnets placed perpendicular to the
flow of the sample. With using a flow chamber with flow path
cross-section 0.5 mm.times.20 mm (h.times.w) and 7 bar NdFeB
magnets, a flow rate as high as 5 mL/min was achieved. After
flowing the mixture through the channel in the presence of the
magnet, a wash solution including heparin was flowed through the
channel. The bound targets were washed with heparin-containing
buffer one time to remove blood components and to reduce formation
of magnetic particle aggregates. In order to effectively wash bound
targets, the magnet was removed and captured magnetic material was
resuspended in wash buffer, followed by re-application of the
magnetic field and capture of the magnetic material in the same
flow-through capture cell.
Removal of the captured labeled targets was possible after moving
magnets away from the capture chamber and eluting with flow of
buffer solution.
* * * * *
References